Resolving the Three-Dimensional Rotational and Translational Dynamics of Single Molecules Using Radially and Azimuthally Polarized Fluorescence

Abstract

We report a radially and azimuthally polarized (raPol) microscope for high detection and estimation performance in single-molecule orientation-localization microscopy (SMOLM). With 5000 photons detected from Nile red (NR) transiently bound within supported lipid bilayers (SLBs), raPol SMOLM achieves 2.9 nm localization precision, 1.5° orientation precision, and 0.17 sr precision in estimating rotational wobble. Within DPPC SLBs, SMOLM imaging reveals the existence of randomly oriented binding pockets that prevent NR from freely exploring all orientations. Treating the SLBs with cholesterol-loaded methyl-β-cyclodextrin (MβCD-chol) causes NR’s orientational diffusion to be dramatically reduced, but curiously NR’s median lateral displacements drastically increase from 20.8 to 75.5 nm (200 ms time lag). These jump diffusion events overwhelmingly originate from cholesterol-rich nanodomains within the SLB. These detailed measurements of single-molecule rotational and translational dynamics are made possible by raPol’s high measurement precision and are not detectable in standard SMLM.

Six-Dimensional Single-Molecule Imaging with Isotropic Resolution using a Multi-View Reflector Microscope

“…Fluorescent molecules are well-approximated as oscillating dipole emitters 29 (Figure 1b), and we model their rotational diffusion as uniform within a hard-edged cone using an orientation vector µ = [ µ x , µ y , µ z ] = [sin θ cos ϕ , sin θ sin ϕ , cos θ ] and solid angle Ω ∈ [0, 2 π ]. To measure molecular orienta-tion, our raMVR approach takes inspiration from both polarization modulation 12,17,30 and pupil-splitting 25,26 innovations in SM orientation-localization microscopy (SMOLM). First, the polarized radiation pattern of a dipole emitter may be manipulated to measure its orientation; 31–33 here, we use a vortex (half) waveplate and polarizing beamsplitter (PBS) (Figures 1a) to separate light from emitters oriented parallel to versus perpendicular to the optical axis.…”

“…This strategy yields isotropic 3D spatial resolution, with raMVR achieving ~26-59% smaller standard deviations on average for measuring the 3D positions and orientations compared to recent, state-of-the-art methods. [11][12][13]15,17 We believe the raMVR pupil-splitting approach has many exciting possibilities for further development. It can be adapted for real-time orientation-resolved epifluorescence microscopy (Figure S14) of cellular structures.…”

“…A variable waveplate (VaWP, Thorlabs LCC1223-A, Figure 1a) and a vortex half waveplate (VWP, Thorlabs WPV10L-633, Figure 1a) are placed before and at the back focal plane (BFP), respectively, in the first 4f system (L1, L2). They convert radially and azimuthally polarized light to x -and y -polarized light, compensating for the birefringence of the dichroic mirrors used in our experiments 17,30 and are calibrated and aligned according to the procedure reported previously. 17 A polarizing beamsplitter (PBS, Meadowlark Optics BB-100-VIS) is placed in the second 4f system (L3-L5, Figure S4a) to create separate imaging channels containing radially or azimuthally polarized fluorescence collected from the sample.…”

“…We follow previously reported procedures 16,17,22 for preparing lipid-coated silica spheres. First, we prepare large unilamellar vesicles (LUVs) composed of DPPC and 40% cholesterol.…”

“…Limited by challenging signal-to-background ratios (SBRs) in typical single molecule (SM) experiments, microscopists have developed various SMOLM techniques to improve measurement performance, 18 approaching the theoretical limits under ideal conditions. [19][20][21] To use precious fluorescence photons more efficiently and maximize signal-to-noise ratios (SNRs), most recently developed techniques create dipolespread functions (DSFs), or images of dipole emitters, with compact footprints 11,12,15,17,22 and thusly achieve higher precision and accuracy. One key challenge with these designs is that orientation estimation requires fitting subtle features within noisy images.…”